Regulator of G-protein Signaling 10 (RGS10) regulates inflammatory signaling pathways in microglia and is implicated in multiple inflammatory disease states. However, the mechanism by which RGS10 controls microglial cell physiology and pathogenesis are undefined. The known biochemical function of RGS10 is to regulate G-protein signaling downstream of G-protein coupled receptors, but this mechanism does not account for RGS10's effects in microglia. RGS10 is enriched in the nucleus of microglia, and it regulates transcription of pro-inflammatory cytokines. Preliminary data suggest that RGS10 functions to regulate transcription by a novel, unknown nuclear mechanism involving indirect chromatin interaction. This represents an unexpected new paradigm in RGS-domain function. However, the specificity of RGS10-DNA interactions and identity of nuclear proteins that mediate the interaction with DNA are unknown. We hypothesize that RGS10 regulates proinflammatory cytokine production by interactions with transcription factors or transcription factor binding proteins in promoter regions of proinflammatory cytokine genes. The objectives of this study are to test this hypothesis, and to generate new testable hypotheses about the physiologic role of RGS10 by fully defining RGS10 protein and DNA interactions in the nucleus of microglia. Specifically, nuclear RGS10 interactions with protein and DNA will be analyzed using unbiased proteomics and next generation sequencing approaches in microglia in the following aims: Aim 1. Define specific DNA sequences associated with nuclear RGS10 in resting and activated microglia. Chromatin Immunoprecipitation followed by next generation sequencing will be used to define specific target sequences associated with nuclear RGS10 and determine whether the profile of target sites is altered following microglial activation. Sequences implicated by unbiased approaches will be confirmed by direct ChIP-PCR. Aim 2. Define RGS10 interactions with nuclear proteins in resting and activated microglia, and determine whether these interactions mediate RGS10 association with DNA. RGS10 co-immunoprecipitation will be performed in nuclear protein extracts from resting and activated microglia, and proteomics analysis of the enriched proteins using LC/MS will identify the nuclear RGS10 interactome. Protein sequences identified in the proteomics study will be confirmed by a series of direct biochemical analyses. Finally, the role of specific RGS10 protein binding partners in mediating the RGS10- DNA interactions defined in Aim 1 will be determined by performing ChIP-PCR following knock-down of specific protein binding partners. The outcome of the proposed project will be to define the first nuclear interactome for an RGS protein and identify for the first time specific sites of RGS protein-chromatin interaction.